Divergent Chemotactic Sensing in Acanthamoeba Reveals Ligand-Promiscuous, Threshold-Tuned Pattern Recognition Without Canonical Formyl Peptide Receptors

Abstract Chemotaxis, the directed movement of an organism towards nutrients or away from noxious agents is a fundamental process for the survival of many microorganisms. We combined high-resolution imaging, microfluidic gradients, and frame-by-frame tracking to re-evaluate Acanthamoeba chemotaxis to microbial glycans (mannan, mannose, N-acetyl-d-glucosamine (GlcNAc), N-acetyl-muramic acid (MurNAc)) and peptides (N-formyl methionyl-leucyl-phenylalanine (fMLP) and Boc-Phe-Leu-Phe-Leu-Phe (BOC-FLFLF)). Our quantitative tracking results on A. castellanii confirm the core patterns in the original studies reported by Schuster and Levandowsky; attraction to fMLP and GlcNAc and lack of response to MurNAc or the peptide antagonist BOC-FLFLF, while revealing previously missed attraction to mannan. In contrast, A. polyphaga demonstrated a more restricted response, with significant chemotaxis observed only toward fMLP, and lack of motility in the presence of MurNAc or BOC-FLFLF. Notably, formyl peptide responses were differentially modulated: BOC-FLFLF reduced fMLP-induced directionality in A. castellanii without impairing motility, while in A. polyphaga, it suppressed both velocity and orientation. When considered alongside genomic analyses that do not reveal a canonical metazoan-like formyl peptide receptor, these behavioural differences suggest that formyl peptide sensing in Acanthamoeba relies on a divergent, pattern-recognition–like signalling strategy, rather than a conserved FPR homolog. These distinct chemoattractant “signatures” are consistent with micro-niche adaptation, and we hypothesise that fine scale tuning of receptor thresholds to local prey spectra contributes to the observed differences between the tested strains. By revisiting classical paradigms, this study offers new perspectives on Acanthamoeba chemotaxis and supports emerging models of protist pattern recognition paralleling innate immunity.